Jeffrey A. Jankowski

Secretion of Catecholamines from Individual Adrenal Medullary Chromaffin Cells

Abstract: Catecholamine secretion has been measured with electrochemical techniques from isolated, single adrenal medullary chromaffin cells with carbon‐fiber microelectrodes. The electrode tip, which is of similar dimensions to the cell, is placed adjacent to the cell to enable the measurement of local secretion. Secretion is caused by exposing the cell to nanoliter volumes of solution containing nicotinic receptor agonists or depolarizing agents. The identification of secreted substances is made with cyclic voltammetry at both bare electrodes and electrodes coated with a perfluorinated cationexchange polymer. Catecholamine secretion is induced by nicotine (10–500 μM), carbamylcholine (1 mM), and K+ (60 mM). All agents that induce secretion lead to a broad envelope of secreted catecholamines on which sharp concentration spikes are superimposed. The concentration spikes can be monitored with a time resolution of tens of milliseconds when the electrodes are used in the amperometric mode. Release induced by nicotine and K+ is inhibited by Cd2+ (0.5 mM), and hexamethonium selectively blocks the nicotineinduced secretion. The actions of nicotine are found to continue for a longer period of time than those of the other secretagogues tested.

Assaying single cells with capillary electrophoresis

Abstract Microseparation methods have become a valuable tool for the analysis of individual cells and cellular subsections. This article reviews the application of capillary electrophoresis to single cell studies by several research groups, emphasizing the variety of detection schemes and sampling protocols developed for assaying such complex microenvironments.

Extracellular Ionic Composition Alters Kinetics of Vesicular Release of Catecholamines and Quantal Size During Exocytosis at Adrenal Medullary Cells

Abstract: The temporal resolution of carbon‐fiber microelectrodes has been exploited to examine the plasticity of quantal secretory events at individual adrenal medullary cells. The size of individual quantal events, monitored by amperometric oxidation of released catecholamines, was found to be dependent on the extracellular ionic composition, the secretagogue, and the order of depolarization delivery. Release was observed with either exposure to 60 mM K+ in the presence of Ca2+ or exposure to 3 mM Ba2+ in solutions of different pH, with and without external Ca2+. Ba2+ was demonstrated to induce Ca2+‐independent exocytotic release for an extended period of time (>4 min) relative to release induced by K+ (∼30 s), which is Ca2+ dependent. In all cases, simultaneous changes of intracellular divalent cations, monitored by fura‐2 fluorescence, accompanied quantal release and had a similar time course. Exocytosis caused by Ba2+ in Ca2+‐free medium had a larger mean spike area at pH 8.2 than at pH 7.4. When Ba2+‐induced spikes measured at pH 7.4 were compared, the spikes in Ca2+‐free medium were found to be broader and shorter but had the same area. Release induced by K+ after exposure to Ba2+ was comprised of larger quantal events when compared with preceding K+ stimulations. Finally, spikes obtained with Ba2+ exposure at an extracellular pH of 5.5 had a different shape than those obtained in more basic solutions. These changes in spike size and shape are consistent with the interactions between catecholamines and other intravesicular components.

Charge-coupled device based fluorescence detection in capillary electrophoresis

Abstract Capillary electrophoresis (CE) is currently one of the highest efficiency small volume separation methods available, and laser induced fluorescence (LIF) detection the most sensitive CE detection method. The design and characteristics of a unique charge-coupled device based CE/LIF system are described that acquires simultaneous fluorescence emission spectra from components separated by CE. The limit of detection for this multichannel detection system is below 10-13 M (less than 100 molecules) for sulforhodamine 101. The advantages of obtaining the wavelength resolved data as opposed to more conventional single channel data are outlined. Two methods for enhancing the ability to attach a fluorescent tag to low concentration peptide samples are presented, including the fabrication of a preconcentrator/reactor at the inlet tip of the capillary and the use of a UV laser with the fluorogenic derivatising agent fluorescamine.